1. Genetic and Physical Mapping Lecture 2

2. Why map genes? Many diseases are partially genetic – Also: environmental factors, randomness 2. We want to identify these genes – Early diagnosis for abortion or regular checks – First step towards developing treatment 3. Individual sequencing is too costly (today) – Sequence a small number of markers – Analyze statistically via biological principles

4. based on the use of genetic techniques to construct linkage maps showing the positions of genes and other sequence features in a genome

5. Physical mapping:

10. Genetic Linkage Maps A genetic linkage map shows the relative locations of specific DNA markers along the chromosome. Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker Markers can be expressed DNA regions (genes) or DNA segments that have no known coding function but whose inheritance pattern can be followed. DNA sequence differences are especially useful markers because they are plentiful and easy to characterize precisely Markers must be polymorphic to be useful in mapping; that is, alternative forms must exist among individuals so that they are detectable among different members in family studies Polymorphisms are variations in DNA sequence that occur on average once every 300 to 500 bp. Variations within exon sequences can lead to observable changes, such as differences in eye color, blood type, and disease susceptibility

11. Stages of Mapping a Gene • Demonstrate disease is hereditary – Show it runs in families • Linkage analysis to identify region – Widely-spaced markers, e.g. RFLPs • Association analysis to narrow region – Closely-spaced markers, usually SNPs • Clone the gene within found region – Investigate its metabolic relevance

15. RAPD

16. SNP

17. AFLP

19. fingerprinting

20. chromosome walking,

24. Types of DNA Markers 1. Restriction fragment length polymorphisms (RFLP) 2. RAPD-Random amplified polymorphic DNA (RAPD) 3. Microsatellite, simple sequence repeat (SSR) markers or short tandem repeat (STR) 4. Single nucleotide polymorphisms (SNPs) 5. Amplified fragment length polymorphism (AFLP)

27. Minisatellites

28. Variable number of tandem repeats = VNTR

29. High number of alleles

30. High level of heterozygocity

31. Problem: southern blots and radioactive probes, minisatellites are to long to be amplified by PCR

32. Not evenly spread over the genome

33. Microsatellites

35. Minisatellites

36. Microsatellites

37. Standard tools for mapping studies and linkega analysis

38. Mostly (CA)n repeats

39. Tri-en tetranucleotide repeats replace more and more the dinucleotide repeats, because of the better results

40. Dinucleotide-repeats are volnurable to “replication slippage” during PCR as such that each allel can give a small ladder on a gel

41. Development of multiplex PCR reactions

50. Physical Mapping Methods Optical mapping 2. Restriction fragment fingerprinting 3. Chromosome walking 4. Sequence tagged site (STS) mapping 5. Fluorescent in situ hybridization (FISH)

53. The digested DNA is labeled with radioactive or fluorescent dye and run on a sequencing gel

54. The fingerprint data is collected and analyzed for contigassemblyDisadvantages: labor intensive and difficult to fill gaps.

59. Clones hybridizing with the same single copy marker are considered to be overlapping

64. PCR confirmation of STS markers in the genome Each STS contains a unique sequence

69. Probes specific to chromosome regions 1p34– 35 and 1p36 were labeled using the ULYSIS Oregon Green 488 (U21659) and Alexa Fluor 594 (U21654) Nucleic Acid Labeling Kits, respectively.(http://www.probes.com/servlets/photohigh?fileid=g001276)

70. FISH mapping of BACs (bacterial artificial chromosomes)

71. Application of FISH in physical mapping Jackson S. et al. 2000. Genetics, 156: 833-838

79. Mice,

80. Drosophila,

81. Caenorabditiselegans , Arabidopsis thaliana

83. A dozen other bacteria have been completely sequenced and all their genes identified, although the functions of most are unknown.

84. Major progress has been made in mapping human genes, and a "rough draft" of the human genome was read by 2000.